Hydrogen sulfide

Volcanic gas

Hydrogen sulfide (H₂S), or just "sulfide" is a microbe-derived off flavor. It is the most common of a group of fermentation products known as volatile sulfur compounds (VSCs). Sulfide aroma and flavor is often described as sulfurous like rotten eggs, "rhino farts", sewer, or volcanic gas. It is also sometimes called a "reductive" aroma because it is more likely to accumulate under low-oxygen conditions.

Sulfide is one of the most common off flavors that occurs in wine and cider. It can also occur in beer and other fermented beverages. In fact, a slight note of sulfide may be acceptable in some styles of lager. The recognition threshold of sulfide is about 1-2 µg/L (parts per billion) and even lower levels can play a role in aroma complexity, or mask desirable aromas.

The odor threshold is well below the threshold for human toxicity, and therefore safety is generally not a concern.

Sulfide should not be confused with sulfite or sulfate.

Formation

Yeast produce hydrogen sulfide naturally, as part of the production of certain amino acids. Specifically, sulfide is an essential metabolic intermediate in the biosynthesis of cysteine and methionine, which are necessary for protein synthesis and cellular metabolism. Sulfide is also now recognized as having important functions in detoxification, population signalling, and extending life span in yeast.

Sulfide is produced mainly by molecular reduction of sulfate or sulfite present in the juice or wort. Sulfate is fairly ubiquitous, and sulfite is a common addition in wine and sometimes in beer. Sulfide is also formed from elemental sulfur, which is sometimes used as an antifungal treatment on grapes. Utilization of these sulfur-containing compounds to produce amino acids occurs through a series of steps called the Sulfate Reduction Sequence (SRS).

Bacteria can also produce sulfide.

Causes of Overproduction

• Yeast strain is one of the main factors influencing the production of sulfide. Some strains of yeast are biologically much more prone to over-producing sulfide.
• Lack of adequate yeast nutrients is another main factor. In order for the yeast to scavenge the sulfide and incorporate it into cysteine and methionine, the yeast need plenty of nitrogen and co-factors such as pantothenic acid to form the precursors for these sulfur-containing amino acids. If there is not enough of the precursor, the yeast release the hydrogen sulfide into the wine or beer.
• Any factors that increase nutrient demand may also lead to increased sulfide production. Pitching an inadequate amount of yeast or an unhealthy yeast culture may cause numerous fermentation-related problems.
• The presence of metals (e.g. copper) during fermentation can stimulate sulfide production.
• Addition or over-use of sulfite may increase or cause sulfide production, particularly with yeast strains used for beer. This is because sulfite is the direct precursor to hydrogen sulfide in the SRS.

Wine only:

• Elemental sulfur is frequently sprayed in the vineyard to fight powdery mildew, and residual sulfur on grapes has been observed to contribute to the formation of sulfide during fermentation by yeast, and the reappearance of VSCs after bottling.

Timing of its Appearance

Maximum amounts of sulfide are liberated when the depletion of nitrogen occurs during the exponential growth phase. Conversely, when depletion of nitrogen occurs during the stationary phase, sulfide liberation is a lower amount and is short-lived. (See Yeast for more about growth phases.)

While sulfide formation occurs mainly during primary fermentation, additional VSCs can be formed at later stages of production, particularly in wine. This phenomenon is rarely a problem in beer production. VSC formation in wine can be difficult to predict and is not necessarily related to sulfide issues during the primary fermentation. The VSCs involved include mercaptans (AKA thiols or mono-sulfides or higher sulfides) and disulfides that have distinctive aromas such as skunky, rubbery, garlic, onion, or cabbage-like. These compounds result from degradation of sulfur-containing compounds in the yeast lees, and chemically-bound sulfide may be released during aging or storage. Sulfide formation has also been reported to occur in the bottle when naturally bottle carbonating with yeast. Even VSCs that had apparently been removed may reappear if conditions in wine become more reductive (e.g. during barrel aging or in the bottle).

There is not always correlation between total sulfide produced by yeast during fermentation and the sulfide concentration in the final wine/beer/etc.

Prevention

It's far better to focus on minimizing the production of sulfide rather than allow it to occur and remove it later because prevention strategies are easier and less damaging/risky than the available options for removing sulfide from the final beverage. For best results, a multi-faceted approach is needed.

For wine and beer:

• Sulfite - The presence of sulfite at the beginning of fermentation has been shown to cause formation of H₂S. Brewers that use sulfite in the wort (i.e. low oxygen brewers) need to adequately aerate/oxygenate the wort to neutralize the residual sulfite when pitching. For wine stabilization, it is recommended to wait at least two weeks before adding sulfite after fermentation ends, particularly when the yeast is still present. Because there is such a high risk of H₂S production when adding sulfite soon after fermentation completes, we do not recommend adding sulfite to beer.
• Aeration - Adding oxygen before pitching yeast is especially important in affecting nitrogen utilization and fermentation vigor, which increases the amount of sulfide stripping from the wine or beer. In wine production, aerating during fermentation is also helpful. (See Aeration)
• Vitamins - Vitamins should be supplemented in wine. Supplementation is not strictly necessary in beer production since wort typically contains adequate vitamins, although it is potentially helpful. Deficiencies of pantothenic acid and pyridoxine (co-factors to SRS enzymes) may cause overproduction of H₂S — even when adequate nitrogen is present. (See Yeast Nutrition)
• Nitrogen - Supplementing yeast-assimilable nitrogen (YAN) can help lower sulfide production, but only when there are also adequate co-factors (vitamins) present for the SRS. Otherwise nitrogen supplementation may increase sulfide production. There may also be some variability among yeast strains or species with regard to whether increasing nitrogen decreases sulfide formation. Similar to vitamins, nitrogen supplementation in wort or beer is not always required, but still may be helpful under certain conditions. (See Yeast)
• Yeast strain - Low sulfide-producing and/or low nitrogen-requirement yeast strains may be considered. Unfortunately it is not very well known which strains are high or low producers of H₂S. Scott Labs and Renaissance Yeast have both bred some wine yeast strains specifically to reduce sulfide production.
• Pitch rate and yeast health - Pitch healthy yeast at a good pitch rate to decrease nutrient demand. "Shocking" the yeast (rapid changes in growth conditions like temperature or pH) should be avoided. Significant over-pitching may also cause excessive sulfide. Even re-pitching yeast may cause increased production of sulfide. (See Yeast)
• Fermentation temperature - Generally lower temperatures decrease sulfide liberation, although not necessarily because of decreased production. However each strain has an optimum fermentation temperature to minimize its production, so lower temperature doesn't always mean lower sulfide production. The fermentation temperature should be controlled within the suggested range of the yeast. (See Temperature control)
• Fermentation duration - A shorter fermentation decreases the amount sulfide ultimately present. This is probably because fermentation time is linked to aeration and nutrient supplementation. (See Yeast)
• Yeast contact - The exact role of lees on sulfide formation has not been established. Aging on lees could be the cause of sulfur-like off flavors, but also the solution to removing them. Evidence of both the release of VSCs from lees and the removal of VSCs by lees has been widely reported. The conditions under which each phenomenon occurs is a very complex matter closely related with the yeast strain and other conditions. For wine, many experts suggest that if the lees start to form these compounds, the wine should be immediately racked. Some experts suggest that only the gross lees are problematic, while the light lees are beneficial. For beer, the absorption of sulfide by yeast has been demonstrated, and home brewers do not report VSC production from aging on yeast.

For wine only:

• Sulfur Spray - Farmers using a sulfur spray should limit residual sulfur on fruit to 7 mg/kg or less (with less than 1 mg/kg being ideal). Stop spraying at least 5 weeks pre-harvest for the lowest risk of sulfide formation.
• Must Clarification - Winemakers can minimize the formation of excess sulfide production in white wines by either settling, centrifuging or filtering the must before fermentation, which removes high-density solids which might contain elemental sulfur. In other words, must clarification will reduce the formation of VSCs. Be aware that fining must with bentonite can remove some nitrogen, which will need to be added back.

Screening

Before attempting to remove sulfur-like off aromas, it's important to know which compounds are present because they are removed with different methods. Any sample checked for reduced aromas must be clear for a valid test.

Materials:

• Tasting glasses
• A measuring device capable of allotting about 50 mL, such as a graduated cylinder or beaker
• A small syringe or pipette to measure 1 mL volumes
• Plastic wrap or watch glasses
• 1% Copper sulfate (CuSO₄) solution (If you can't obtain copper sulfate, you can clean several U.S. pennies in an acid solution like lemon juice or vinegar.)
• Optional: Ascorbic acid and distilled water to make a 5% ascorbic acid solution. Mix 2.5 g ascorbic acid into 50 mL water.

Procedure:

1. Obtain two 50mL samples of wine in glasses. Label one "Control" and the other "Copper".
2. To the glass marked "Copper", add 1 mL of the copper sulfate solution (approx 50 ppm - this is a strong excess of copper), or pennies.
3. Cover both glasses with a watch glass or plastic wrap and swirl.
4. Let glasses sit for around 15 minutes and then examine by smell. Do NOT taste experimental glass.

Determining the results:

1. If the off odor is gone from the experimental glass, it is likely that only hydrogen sulfide and/or mercaptans are present.
2. Otherwise, if the experimental glass is still stinky, this can mean that the odors are disulfides and/or dimethyl sulfide (DMS), neither of which react with copper.
3. In the second case, you need to differentiate between disulfides and DMS. Create a third glass with a 50 mL sample labeled "Copper and AA". To this glass add 1 mL of 5% ascorbic acid solution 5 minutes before adding 1 mL copper sulfate solution. Follow the same evaluation procedure. If the sulfur-like off aroma is removed from this glass, it means disulfides are present. Ascorbic acid only works in a sample with adequate sulfite levels (30ppm Free SO₂ or higher).

Removal

While large amounts of sulfide may be produced during fermentation, much of this sulfide is usually volatilized (off-gassed) from the wine or beer along with CO₂ during active fermentation. Therefore, these removal methods should only be applied after fermentation is complete.

These removal procedures will cover the 3 types of VSCs that cause off-flavors: hydrogen sulfide, mercaptans, and disulfides. For best results, conduct a screening procedure before making interventions because some interventions make impair the removal of certain VSCs under certain circumstances.

Almost all of these methods have a potential negative effect, which is what makes prevention so important.

Yeast contact

Increased yeast contact after fermentation may help remove all types of VSCs. Wine may benefit from racking off the gross lees. See the Prevention section above for more discussion.

Oxidation of desirable compounds is the only potential negative effect of increased time in the primary fermentation vessel.

Sparging with inert gas

Hydrogen sulfide is highly volatile. If you have the appropriate equipment, it can be removed through "sparging" with inert gas (such as nitrogen or carbon dioxide). In other words, bubbling gas through the beer/wine/etc will carry off the hydrogen sulfide along with it.

Perform this method only in a well-ventilated space. The specifics of a gas sparging setup are beyond the scope of this article, but you will need a gas cylinder, an appropriate regulator, gas tubing, and an "oxygen stone".

This approach is less effective against mercaptans and disulfides. It will also strip desirable aromatic compounds.

Oxygen exposure

Hydrogen sulfide is easily oxidized to elemental sulfur, which is insoluble and flavorless. If fermentation is still active, stirring it may help volatilize and/or gently remove sulfide with low risk of also oxidizing desirable compounds. If fermentation has completed, you can simply leave the beer/wine/etc in the fermenter and oxygen that enters the vessel will react with hydrogen sulfide. Aeration (e.g. through splash racking) may also be used, particularly in wine with sulfite.

Oxygen exposure does not remove mercaptans or disulfides. Furthermore, most sources suggest that aeration adds a danger of forming mercaptans and/or converting mercaptans to disulfides. The disulfides have a higher taste threshold so they may seem to disappear, but they can potentially change back to mercaptans later under low-oxygen conditions such as a wine with sulfite. This phenomenon does not occur in beer because beer always becomes increasingly oxidized over time.

Oxygen exposure (including aging without the anti-oxidant protection of sulfite) can cause oxidation of desirable compounds, which negatively affects flavor.

Copper

Copper is a common tool used for the removal of both hydrogen sulfide and mercaptans. Copper binds to these sulfur compounds to form odorless complexes, which precipitate to some degree. When copper is used in combination with ascorbic acid and sulfite, disulfides can be removed as well (see below). Adding copper is fairly easy and inexpensive. Copper fining works in both wine and beer.

Copper should not be added until the fermentation is complete and the amount of yeast material is reduced by racking. This is because yeast cells can bind with copper and reduce effectiveness, and because addition of copper during fermentation may promote sulfide production by yeast. However some yeast may be helpful for removing the excess copper. The beer/wine should be left undisturbed for several days after treatment so the copper sulfide (a very fine black powder) can settle to the bottom of the container. Then it should be carefully racked off the residue.

Despite its widespread use, copper usage has a lot of potential disadvantages. The copper-sulfide complexes are challenging to remove from wine/beer, and they can potentially release the sulfide or mercaptans later. Copper might catalyze the release of sulfide from sulfur-containing amino acids. Copper also reacts with any other thiols in the beer/wine. Therefore, if you are dealing with a wine variety rich in aromatic varietal thiols (e.g., Sauvignon blanc, rosés, and to a lesser extent Riesling and Gewürztraminer), the addition of copper can reduce the wine's varietal aroma. In beer, thiol-containing hop compounds may be affected. Too much copper can cause a haze, referred to as "copper casse". Risk of haze formation is greatly increased if copper is added immediately prior to packaging, without allowing adequate time for the beer/wine to stabilize during bulk storage. Lastly, excess copper catalyzes oxidation reactions, which can accelerate staling. It's even possible that copper additions may actually increase the amount of VSCs in the final wine!

For those reasons, the best results are obtained by using the minimum amount of copper needed to remove the offensive VSCs. To do this, a "bench trial" should be performed to determine the minimum effective amount. Old-fashioned methods such as stirring with a copper pipe should be avoided because that practice may lead to excessively high levels of copper. If a bench trial is too complicated, you may add copper sulfate directly to the wine in incremental amounts (0.05-0.1ppm at a time). However that may end up being more work in the long run.

Bench trial materials:

• Glasses, vials or flasks, 50 mL or larger (although closer to 50 mL is preferred)
• A measuring device capable of allotting about 50-100 mL, such as a graduated cylinder or beaker
• A small syringe or pipette to measure 0.1 mL volumes
• Plastic wrap, stoppers, or watch glasses
• Distilled water
• 1% copper sulfate solution

Bench trial procedure:

1. Create a 0.01% copper sulfate solution by adding 1 mL of the 1% copper sulfate solution to a beaker or graduated cylinder and topping it up to 100 mL with water.
2. Put 50 mL samples into four glasses.
3. Label the following glasses: (1) control, (2) 0.1 ppm copper, (3) 0.2 ppm copper, and (4) 0.5 ppm copper.
4. Using a 0.01% copper sulfate solution, add the following increments to each glass:

   0.0 mL to glass 1 = no copper addition

   0.2 mL to glass 2 = 0.1 mg/L copper addition

   0.4 mL to glass 3 = 0.2 mg/L copper addition

   1.0 mL to glass 4 = 0.5 mg/L copper addition

5. Cap each glass and wait 12-24 hours.
6. Sniff (smell only!) each glass to determine when the offensive aroma is gone. This is the concentration of copper sulfate that will need to be added to the beer/wine. (0.04 mL of 1% copper sulfate per liter of beer/wine gives 0.1 mg/L copper.)

Notes:

• Try to minimize headspace in the sample glasses, as air that can cause variation through oxidation.
• Feel free to alter the concentrations evaluated and/or conduct additional trials. As little as 0.05 mg/L may be needed, and the maximum copper should not exceed 6 mg/L.

Small amounts of excess copper sulfate (between 0.1-0.2 mg/L) can be removed with bentonite, yeast hulls, or fresh lees additions. However, the majority of added copper remains in wine and is not readily removed by racking or filtration.

Other copper products:
Copper citrate may be a good alternative to copper sulfate because supposedly copper citrate does not totally go into the ionic form, and therefore does not leave as much residual copper in the wine.

Kupzit® contains 2% copper citrate. For easy dosage and handling, it is coated onto a mineral carrier material, a particularly pure, high-quality granulated bentonite.

• 1g copper sulfate pentahydrate contains 0.255g copper
• 1g copper citrate hemipentahydrate contains 0.353g copper
• 50g Kupzit contains 1g copper citrate, which contains 0.353g copper

Another alternative product is Reduless®, which is a proprietary fining product from Lallemand that can "naturally enhance roundness while treating sulfur problems and reducing phenol related defects". However, their claim that it removes DMS is pretty unlikely. Being proprietary, they don't really disclose what's in it, but it includes bentonite, inactivated yeast, and "natural elements which are rich in copper". Reduless® may also be easier to handle compared to copper sulfate solution.
Suggested usage is 0.10-0.15 g/L, which adds no more than 0.02 mg/L copper (a low amount). If using this product, the copper sulfate bench trial described above isn't helpful since there's not a dosage conversion to determine how much Reduless would be needed to achieve the same effect.

Ascorbic acid

Ascorbic acid enables the removal of disulfide compounds by converting them to mercaptans, which allows copper to bind with them.

First, it's important to make sure that free SO₂ levels are at least 30 mg/L before adding ascorbic acid. Otherwise ascorbic acid will not help, and it will potentially lead to oxidation. (See Sulfite and SO2 testing)

Ascorbic acid in the range of 20-100 mg/L may be used, depending on how strong the off odor is and how well it seemed to respond in the screening evaluation above. Many sources suggest starting with around 50 mg/L or higher. However, the well-respected Australian Wine Research Institute (AWRI) recommends adding much lower levels: For white wines, add 10 mg/L ascorbic acid and then another 10 mg/L the following day. For red wines, add 2 mg/L ascorbic acid and then another 2 mg/L the following day. However those recommendations are for commercial wine, which may have lower levels of disulfides compared to a problematic home brew.

To add ascorbic acid, simply calculate the amount based on your target level, dissolve it in a bit of water, and then add to your wine or beer.
Here's an example calculation to target 50 mg/L for 20 L of wine:
50 * 20 ÷ 1000 = 1 gram of ascorbic acid
For lower levels or smaller volumes, it may be wise to make a stock solution as described in the screening procedure so that measurements will be more accurate.

After the ascorbic acid addition, we need to give the chemical reactions enough time to occur before conducting a copper bench trial (or adding copper to the batch). Unfortunately this process can take days to months, and recommendations for how long to wait are inconsistent. The AWRI suggests to wait only 24 hours after the second addition of ascorbic acid. Other sources suggest to wait about 4-5 days, or as long as several weeks. If the subsequent copper bench trial is unable to fully remove the odor with reasonable amounts of copper, consider adding more ascorbic acid, or giving it more time.

Ascorbic acid can trigger the release of sulfides and mercaptans from the copper. Therefore, the lowest effective amount of ascorbic acid may be preferred, so as not to interfere with the action of copper. However, given the slow speed of reaction, and the fact that it doesn't improve the odor by itself, bench trials to determine the lowest effective amount are not practical. The other potential downside of using ascorbic acid is that it can lead to faster oxidation if the level of sulfite is too low.

Note that there are many sulfide compounds that do not respond to fining with ascorbic acid and copper. Examples of these include diethyl sulfide (DES) and dimethyl sulfide (DMS). Unfortunately, there is no good or predictable way for us to remove these compounds.

Other measures

Tannins - Enartis (a company that produces winemaking products) claims that the addition of tannins, especially ellagic (oak) tannins, has the ability to bind with mercaptans and form odorless complexes. These complexes are supposedly very stable over time and do not entail the risk of a later sulfur off-aroma appearance. However, we can't find any scientific research to support this. In fact, one study showed that adding oak does not seem to affect the levels of VSCs, with or without microxygenation (along with adequate sulfite levels). There has been some anecdotal success with 0.1-0.2 g/L chestnut tannins for removing H₂S, but not other VSCs.

Mineral Oil - As crazy as it sounds, disulfides can be removed by adding mineral oil to the wine and agitating it daily for several days. This works because the disulfides are more soluble in oil compared to the wine. When the wine beneath the mineral oil layer smells clean, it should be racked, leaving the oil behind. An additional racking may be needed to fully remove the oil. This may be useful as a last resort for removing disulfides from wine, but for beer it's probably not a great idea.

Silver Chloride - This additive is effective for removal of hydrogen sulfide, mercaptans, AND disulfides, but this compound has limited availability, it is relatively expensive, and there is very little information about its use.

Science

Biology and sulfide formation
For a review of the biological production pathways, see these articles:

• Wang XD, Bohlscheid JC, Edwards CG. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J Appl Microbiol. 2003;94(3):349–359.
• Huang CW, Walker ME, Fedrizzi B, Gardner RC, Jiranek V. Hydrogen sulfide and its roles in Saccharomyces cerevisiae in a winemaking context. FEMS Yeast Res. 2017;17(6).

Sulfite
To be utilized for production of H₂S, SO₂ simply diffuses into the cell, equilibrates at cytosolic pH as bisulfite (HSO₃^-) and sulfite (SO₃^2-), and thereby can actually accumulate to 60-times its extracellular concentration.

Myth busting
Some sources claim that adding sulfite after fermentation theoretically helps eliminate sulfide by reacting with it. However, this appears generally not to be the case in practice, otherwise sulfide would basically never be a problem in wine. Perhaps it is instead related to oxygen exposure. Other sources indicate that H₂S may simply exist in equilibrium with products of a sulfite + sulfide reaction, which also serve as a latent reserve of H₂S that would release as sulfite declines during aging — this is arguably even worse than not helping.

Certain sources (Beer & Brewing) claim that high levels of sulfate in brewing water may lead to increased sulfide production. This isn't supported by any modern science, nor the most modern textbook they reference (Handbook of Brewing, 2006). Jiranek (1992) showed that fermentations containing as much as 4800ppm sulfate (50mM) produce negligible amounts of sulfide.

Chemistry
Reaction of sulfide with oxygen:
2 H₂S + O₂ → 2 S + 2 H₂O

Reaction of sulfide with copper sulfate:
H₂S + CuSO₄ → CuS + H₂SO₄

There is still a great deal of uncertainty with regard to the formation and chemical reactions of both sulfur compounds and copper compounds. For a more thorough explanation of why this is the case, refer to the article by Müller and Rauhut (2018) if you dare.

Signalling
Sulfide has been shown to perform an important signaling function. H₂S will arrest the respiration phase and signal the onset of fermentation. This allows the population of cells to coordinate metabolic activity. H₂S is deliberately made and released to coordinate rapid fermentation onset. There could be a strong selective advantage to the production of H₂S under certain environmental conditions.

Mercaptans and disulfides
The higher sulfide compounds (e.g. mercaptans and disulfides) are believed to largely generate from the degradation of the sulfur-containing amino acids. Spiking wines with methionine, cysteine or the cysteine-containing tripeptide glutathione leads to the formation of these compounds in juices and wines. Some of these compounds appear to continue to increase during storage of the wine after yeast activity has ceased, suggesting that there are precursor forms present in wine that, as the reductive conditions of the wine change, generate VSCs. In beer, for example, dimethyl sulfide (DMS) can form from reduction of Dimethyl sulfoxide derived from S-methylmethionine (SMM). This pathway has not been shown to exist in wine, where dimethyl sulfide is believed to come from the degradation of cysteine, glutathione, methionine or S-adenosyl-L-methionine. Some higher sulfides may also come from degradation of sulfur-containing pesticides, but this is a rare occurrence and more often these compounds are derived from catabolism (break down) of sulfur-amino acids and their derivatives, glutathione and S-adenosyl-L-methionine. Thioalcohols can also be found in wine. These components likewise are believed to derive from degradation products of S-containing amino acids and their derivatives or from the interaction of H₂S with acetaldehyde, which forms the reactive 1,1-ethanedithiol, and other reactive components in wine. Some of these products are reactive themselves, leading to even more diverse VSCs. The chemical reactivity of these compounds in combination with a host of potential reactants in wine and very low thresholds of detection, has made it challenging to delineate the true pathways by which they are formed.

Cysteine
The amino acid cysteine is commonly referenced as contributing to increased formation of hydrogen sulfide, but in practice its effect is negligible since cysteine concentration in must is typically very low. It's believed that aspartate aminotransferase deaminates cysteine to give mercaptopyruvate, which in a subsequent step catalyzed by MST liberates H₂S and pyruvate. However, that hasn't been fully confirmed by other sources.

Methionine
Methionine addition has a variable effect on hydrogen sulfide (and sulfite) production, and the effect also varies by yeast strain.

See also

• Article about VSCs in beer by Randy Mosher.
• Scientific discussion of the variety of VSCs and their formation from UC Davis.
• Copper sulfate calculator from the AWRI.